Establishment and characterization of Epstein-Barr virus gp350-expressing transfected human lymphoid (Raji) cell...

Gp350, a late Epstein-Barr-virus (EBV) glycoprotein expressed on both the envelope of viral particles and EBV-producing cells, is also the candidate for the development of an anti-EBV subunit vaccine. This glycoprotein is thought to play an important role in anti-EBV immunity. However, studies on the role of this viral antigen in cellular cytotoxicity and other immune functions have been hampered by the lack of a suitable model expressing gp350. We describe here a study in which we successfully transfected a gp350-negative cell line resistant to natural-killer(NK)-cell activity (i.e., Raji) with a recombinant plasmid (pZIP-MA) containing the EBV-gp350 and the neomycin resistance gene. Three clones with a stable and strong expression of gp350 on their surface membrane, as demonstrated using a gp350-specific (i.e., 2LI0) monoclonal antibody (MAb) were isolated, characterized and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. However, gp350 expression on 2 of the 3 isolated clones was not recognized by an anti-gp350 MAb (72AI) which is specific to a unique gp350 epitope with a dual function (i.e., involved in both EBV binding to its target cell receptors and in inducing virus-neutralizing antibody). We have also found that gp350 expression on our 3 selected clones does not affect EBV-receptor (CR2) density. Our model of gp350-expressing, NK-cell-activity-resistant targets revealed very useful in determining that gp350 serves as a target antigen for EBV-specific ADCC. These gp350-expressing cell clones appear to represent a valuable tool for diagnostic purposes (i.e., for detecting and titrating gp350 antibodies in patients with EBV-associated diseases). Our approach should also prove useful for studying the expression of other cell-surface-associated viral and tumor antigens and their role in specific cellular immunity and immunosurveillance.